The aims of this project are: (1) to develop and modify NMR methodologies for the assignment of resonances and the analysis of the structure and dynamics of molecules in solution, and (2) to apply these methods to problems of concern in relation to environmental health. During the past year, the primary emphasis of this project has been in two areas: (1) development of the transferred NOE (TRNOE) technique and its application to the determination of the conformation of the nucleoside drug tubercidin in the complex with E. coli purine nucleoside phosphorylase (PNPase), and (2) analysis of the interaction of benzeneboronic acid (BBA) derivatives with small molecules and with the enzyme subtilisin. An important objective of both problems was a characterization of the kinetic behavior. Specifically, we have previously determined that the TRNOE parameters for inhibitors which are exchanging with macromolecular binding sites can be strongly dependent on the exchange rates. We have recently evaluated the use of two strategies for determining ligand exchange rates: (1) Measurement of T2 using the CPMG technique as a function of pulse rate, and (2) measurement of the T1o values for the ligand as a function of the strength of the spin-lock field. We are currently carrying out detailed theoretical and experimental analyses of these experiments in the PNPase - tubercidin system. In the second area, studies with BBA derivatives have determined apparent dissociation constants with various biologically significant ligands under physiological conditions, as well as binding kinetics. The binding kinetics of 4-fluoroBBA with the serine protease subtilisin were compared with results obtained on various model ligands in order to characterize the interaction with the enzyme.